With the recent dramatic rise of antibiotic-resistant pathogens and infectious diseases, the need for new antimicrobial agents is urgent (Cohen et al., 1992, Science 257:1050-1055). For example, strains of Enterococcus faecium that are resistant to vancomycin have recently been observed (Moellering, 1990, Clin. Microbiol. Rev. 3:46-65). As vancomycin is considered to be the antibiotic of last resort for several pathogens, strains resistant to vancomycin pose a serious health threat to society. Despite this urgency, in more than ten years only one completely different type of antibiotic, a streptogramin mixture called Synercid (Rhone-Poulenc Rorer, Collegeville, Pa.), has reached Phase III clinical trials (Pfeiffer, 1996, "New Anti-Microbial Therapies Described," Genetic Engineering News 16(8):1).
Recently, a new class of antimicrobial or antibiotic agents based on naturally-occurring antimicrobial peptides produced within plants, animals and insects have been discovered. These peptides include, among others, cecropins (Hultmark et al., 1980, Eur. J. Biochem. 106:7-16; Hultmark et al., 1982, Eur. J. Biochem. 127:207-217), apidaecins (Casteels et al., 1989, EMBO J. 8:2387-2391), magainins (Zasloff, 1987, Proc. Natl. Acad. Sci. U.S.A. 84:5449-5453; Zasloff et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:910-913), tachyplesins and analogues of tachyplesins such as polyphemusins (Nakamura et al., 1988, J. Biol. Chem. 263:16709-16713; Miyata et al., 1989, J. Biochem. 106:663-668), defensins (Lehrer et al., 1991, Cell 64:229-230; Lehrer et al., 1993, Ann. Rev. Immunol. 11:105-128; U.S. Pat. No. 4,705,777; U.S. Pat. No. 4,659,692; U.S. Pat. No. 4,543,252), .beta.-defensins (Selsted et al., 1993, J. Biol. Chem. 288:6641-6648; Diamond et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:3952-3958), insect defensins (Lambert et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 88:262-265; Matsuyama and Natori, 1988, J. Biol. Chem. 263:17112-17116), and protegrins (Kokryakov et al., 1993, FEBS 337:231-236; Zhao et al., 1994, FEBS Letters 346:285-288; Migorodskaya et al., 1993, FEBS 330:339-342; Storici et al., 1993, Biochem. Biophys. Res. Commun. 196:1363-1367; Zhao et al., 1994, FEBS Lett. 346:285-288; Manzoni et al., 1996, FEBS Lett. 383:93-98; U.S. Pat. No. 5,464,823). The discovery of these new classes of antimicrobial peptides offers hope that some might be developed into agents that can be used against microorganisms of medicinal importance. Those of animal origin are of particular importance, as these antimicrobial peptides generally exhibit activity against antibiotic-resistant bacterial strains and have a lower frequency of resistance as compared to conventional antibiotics (Steinberg et al., 1996, "Protegrins: Fast Acting Bactericidal Peptides," presented at: Intl. Symposium on Staphylococci and Staphylococcus Infections, Aix les Bains, France). At least one of these peptides, called Cytolex.TM. (Magainin Pharmaceuticals, Inc.), is currently in Phase III clinical trials for infections associated with diabetic foot ulcers (Craig, Aug. 17, 1995, BioWorld Today 6(158):1).
One particularly interesting class of antimicrobial peptides are those which have been isolated from porcine leukocytes, designated "protegrins". In addition to the five naturally-occurring protegrins, designated PG-1, PG-2, PG-3, PG-4 and PG-5, respectively, several active congeners have been described (see, e.g., U.S. Pat. No. 5,464,823; WO 95/03325; WO 96/37508). The protegrin peptides, which are generally amphiphilic in nature, exhibit antimicrobial activity against a broad spectrum of microbes, including viruses, retroviruses, bacteria, fungi, yeast and protozoa. In addition, they bind to endotoxins, i.e., the lipopolysaccharide (LPS) compositions derived from Gram-negative bacteria that are believed to be responsible for Gram-negative sepsis. Thus, these peptides are useful as antibacterial, anti-fungal and antiviral agents in both plants and animals. For a review of the literature concerning protegrin peptides, see, Kokryakov et al., 1993, FEBS Lett 337:231-236; Zhao et al., 1994, FEBS Lett 346:285-288; Mirgorodskaya et al., 1993, FEBS Lett 330:339-342; Storici et al., 1993, Biochem Biophys Res Comm 196:1363-1367; Harwig et al., 1995, J Peptide Sci 3:207; Zhao et al., 1995, FEBS Lett 376:130-134; Zhao et al., 1995, FEBS Lett 368:197-202; Miyakawa et al., 1996, Infect Immun 64:926-932; Yasin et al., 1996, Infect Immun 64:709-713; Qu et al., 1996, Infect Immun 64:1240-1245; Aumelas et al., 1996, Eur J. Biochem 237:575-583; Mangoni et al., 1996, FEBS Lett 383:93-98; Steinberg et al., 1996, "Protegrins: Fast Acting Bacterial Peptides," presented at 8th Intl. Symposium on Staphylococci and Staphylococcal Infections, Aix les Bains, France, Jun. 23-26, 1996; Steinberg et al., 1996, "Broad Spectrum Antimicrobial Activity of Protegrin Peptides," presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996; Kung et al., 1996, "Protegrin Protects Mice From Systemic Infection By Antibiotic-Resistant Pathogens," presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996; and Steinberg et al., 1996, "In Vitro Efficacy of Protegrins Against Helicobacter pylori," presented at 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996.
In use, protegrin peptides provide myriad advantages over conventional antibiotics and other antimicrobial peptides. For example, unlike defensin peptides, protegrin peptides effect their broad spectrum activity under physiological conditions, including in the presence of physiological saline. Due to their small size, they can be prepared in non-immunogenic form, extending the number of species to which they can be administered. Moreover, since the protegrin peptides are related to antimicrobial peptides found naturally in animals, they do not exhibit the high frequency of resistance observed with traditional antibiotics. Thus, the protegrin peptides are particularly useful for treating or preventing infections caused by antibiotic-resistant pathogens.
The present invention is directed to a new set of protegrin peptides which offer improved serum compatibility, and hence improved utility as systemic antibiotics, as well as decreased hemolytic activity against human red blood cells as compared with the naturally-occurring protegrins, while at the same time providing broad spectrum activity with a low frequency of resistance.